The use of short-interfering RNAs (siRNAs) to trigger RNA interference (RNAi) is actively being pursued as a therapeutic approach against many human diseases. However, the efficacy of RNAi as a gene-silencing therapy requires understanding how therapeutic siRNAs enter cells to gain access to the silencing machinery. In addition, we must rationally determine how to modify these dsRNAs molecules for more efficient delivery and targeting to select tissues and cells. Studies of dsRNA uptake and silencing in the nematode C. elegans have been instrumental in identifying key proteins involved in the process. Genetic screens for systemic RNAi defective (sid) mutants have identified two transmembrane proteins, SID-1 and SID-2 required for dsRNA uptake. SID-1 is an ancient protein with homologs detected in all sequenced vertebrate and many invertebrate genomes. A human homolog of SID-1 has recently been shown to be critical for cytoplasmic delivery of modified siRNAs, suggesting that dsRNA transport is a conserved function for this family of channel proteins. Current work in our laboratory has shown that knockdown of mouse SID-1 disrupts a wide range of processes, including animal growth, neuronal functions, and defects in immune system development. In contrast, SID-2 appears to be a rapidly evolving protein that is detectable by sequence similarity only in Caenorhabditis nematodes. However, the process that SID-2 enables, silencing by RNA uptake from the environment, is more widespread. Expression of either SID-1 or SID-2 in cells normally refractory to dsRNA, enables dsRNA import. Understanding the regulation and function of these proteins in the experimentally tractable nematode C. elegans and experimental vertebrate systems will provide valuable insights for the development of therapeutic RNAi in humans. The long-term objective of the proposed research is to understand the physiological importance and mechanism of intercellular RNA transport in animals. Towards this end, the specific aims of this proposal are: 1) To characterize the specificity and RNA transport mechanism of the dsRNA channel. 2) To characterize proteins that function with SID-1 and SID-2 and are necessary to allow dsRNA uptake using biochemical and genetic approaches. 3) To investigate the dsRNA channel in mouse immune-system development and function. 4). To isolate and characterize endogenous extracellular RNAs in C. elegans. This work may identify an ancient, gene-specific mechanism for intercellular communication in animals.